Large polyacrylamide hydrogels for large-batch cell culture and real-time tunable stiffness

Introduction:: The rigidity of a cell’s substrate or extracellular matrix plays a vital role in regulating cell and tissue functions. Polyacrylamide (PAAm) hydrogels are one of the most widely used cell culture substrates that provide a physiologically relevant range of stiffness. In preparation of the PAAm substrate, a PAAm hydrogel is typically polymerized between an adhesive or silanized glass coverslip and a non-adhesive hydrophobic glass slide. The polymerized hydrogel is thus physically or covalently attached to a rigid substrate at its bottom surface, and the hydrophobic slide is removed to expose the cell-contacting surface. However, this approach as well as other modified methods is associated with difficulties, including time-consuming for high-yield cell culture, technical and experiences demanding, suffering easily peeled off, and containing mechanical variations. In the first part of this study, we present a simple method to prepare large PAAm hydrogels with less time cost and easily accessible materials. The stiffness of the hydrogel covers a large range of Young’s modulus and regulates cell behaviors, and the hydrogel is mechanically uniform and supports cell culture in a large batch. Based on this method, in the second part of this presentation, we develop a PAAm-based magnetorheological substrate with stiffness that can be tuned rapidly and reversibly in real-time on demand, simply through an application of magnetic field. In sum, the methods we presented here improve reproducibility of mechanobiology studies and can be easily applied for mechanobiology research requiring large numbers of cells or experimental groups.


Materials and Methods:: Our method prepares the large PAAm hydrogel between two polystyrene surfaces with a 100 mm, large polystyrene petri dish. As illustrated in Figure 1, the pre-gel solution containing acrylamide, bis-acrylamide, APS, and TEMED is transferred into a plastic petri dish lid with the opening side facing up. The bottom half of the petri dish, likewise opening facing up, is then placed onto the pre-gel solution covering it. In this way, the hydrogel is sandwiched between the two petri dish surfaces. The magnetorheological hydrogel contains additional 60% w/v magnetizable carbonyl iron particles, which are dispersed inside the polymeric hydrogel network. In the presence of external magnetic field, the inside particles are magnetically polarized within several milliseconds, making them resist hydrogel macroscopic deformation. This feature allows the stiffness of the hydrogel to undergo significantly increase with magnetic field, making the hydrogel stimuli-responsive. After polymerization in a humidity chamber for 30 minutes, the top dish is removed, and the hydrogel is taken out from the dish lid. The resulting hydrogel, except for the out-of-dish boundary region, is transparent, flat with no curling. The hydrogel stiffness was measured by a stress-controlled rheometer with 1% shear strain at 1 Hz. The non-treated petri dish is commercially available and inexpensive. In addition, unlike the glass-sandwiching method which requires hydrophobic and gel-attaching treatments to the glass slides, our method does not involve any surface treatment, as the petri dish is non-plasma-treated and is hydrophobic. Together, our method is cost-effective yet generating a large-sized hydrogel in the meantime.


Results, Conclusions, and Discussions:: We first determine the mechanical uniformity of the large PAAm gels (Figure 2). We find that for each stiffness of formulation, there is less than 5% variance in their elastic shear moduli G’ across one large gel and less than 2.5% variance among three parallel gels. We therefore conclude that the hydrogel is mechanically uniform and experimentally reproducible.

We next study the cell culture capacity of the large hydrogel substrate (Figure 3). We demonstrate that the large PAAm substrates support large-batch cell culture, and By Western blot experiments especially targeting on the low-abundance proteins, the large substrates could produce ample amount of whole cell proteins for protein analyses.

We further ask whether the PAAm hydrogel synthesized by our novel method could be applied as a platform for mechanobiological research (Figure 4). To assess it, we study the cellular responses to hydrogels with different stiffnesses in mouse embryo fibroblasts. After a one-day incubation, cells on stiffer gels exhibit a significantly larger spreading area and a higher growing rate, and are 10 times more contractile compared to cells on softer gels. We thus conclude that our novel hydrogel preparation method can support research in biomechanics and mechanobiology.

Finally, we develop a PAAm-based magnetorheological substrate with stiffness that can be tuned rapidly and reversibly in real-time (Figure 5). We characterize the magnetorheological responses of the synthesized MR gels with a N52 permanent magnet and find the elastic shear modulus (G’) of the hydrogel shows a 18x increase from 370 Pa to 6.8 kPa. The increase in G’ can last for at least two days and is fully reversible upon removal of the permanent magnet. The MR hydrogel showed no observable toxicity to cells, and we find that cells are responsive to the stiffness of the MR hydrogel regulated by the permanent magnet. The ongoing experiment includes studying the time scale of cell responses to time-varying stiffness.

Altogether, we present a much easier and more cost-effective way to generate PAAm hydrogel substrates, which may be highly useful in future studies on cellular forces and other cell responses to substrate stiffness.


Acknowledgements (Optional): : This work was supported by NIH GM136259 award.


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